This application focuses on a globular protein of central medical importance - human proinsulin - and investigates sequence determinants of its foldability. Proper folding of proinsulin is fundamental to the function of the pancreatic beta cell and maintenance of metabolic homeostasis in vertebrates. Little is known about the baseline structure of proinsulin due to long-standing difficulties in obtaining crystals and the intractability of its NMR spectrum due to aggregation. In Aims 1 and 2 we will overcome these limitations through heteronuclear NMR studies of an engineered monomer. We seek to compare the structure of the insulin moiety to that of insulin itself and to evaluate the extent of local order in the connecting peptide. Aim 3 investigates sequence determinants of foldability in the endoplasmic reticulum of mammalian secretory cell lines. These cell-biological studies will test the hypothesis that residues in a specific folding nucleus are required for proper folding independently of effects of mutations on the stability or receptor-binding affinity of the folded protein. We hypothesize that "folding mutations" will be found that block kinetic accessibility to the ground state. Aim 4 builds on these results to test whether putative in vivo folding mutations indeed impair oxidative folding of proinsulin in vitro. Because structural studies of nonfolding variants might be infeasible using biosynthetic material, we propose to employ total chemical synthesis to prepare the corresponding mutant insulins for functional, thermodynamic, and structural studies. Structures of "unfoldable" variants are sought in Aim 5. Can specific side chains in a protein serve as "kinetic guides" during folding - but be dispensible for the fuction of the protein once folded? Classical folding-pathway mutations have been identified in the trimeric tail-spike protein of phage P22. The proposed studies will test whether this paradigm generalizes to a monomeric folding reaction of central pharmaceutical and physiological importance. Of overarching interest would be the crystal structure of an "unfoldable" protein. Our results promise to have implications for the evolution of insulin-like sequences and the possible misfolding of human proinsulin in type II diabetes mellitus.